Angiogenesis, the process by which new capillaries are formed from pre-existing blood vessels, is essential for the growth and persistence of solid tumors and their metastases. Pathogenic angiogenesis plays an important role in the progression of diseases, such as cancer, diabetic retinopathy, psoriasis, rheumatoid arthritis, etc. Under stable conditions, vascular endothelial cells exist in a quiescent state while maintaining a relatively slow turnover. The switch involving the conversion of quiescent endothelial cells to an active pro-angiogenic phenotype requires both the up-regulation of endogenous angiogenesis stimulators and the down-regulation of endogenous angiogenesis inhibitors. Such angiogenesis stimulators may include, for example, bFGF, VEGF, vascular permeability factors, and the like, while endogenous angiogenesis inhibitors may include, for example, angiostatin, endostatin, tumstatin, canstatin, arresten, thrombospondin, and the like.
Among these angiogenesis inhibitors, endostatin is a 20 kDa polypeptide derived from collagen XVIII and an endogenous anti-angiogenesis protein that inhibits endothelial cell proliferation, migration, invasion, tube formation, etc. Endostatin is released from the collagenous domain by cleavage within the protease-sensitive hinge region by enzymes, such as elastase and cathepsin, and circulates in the blood at a concentration of from 20 to 35 ng/ml. Endostatin specifically binds to a specific integrin and inhibits the phosphorylation of focal adhension kinase (FAK). The inhibition of FAK phosphorylation by the binding of endostatin to integrin leads to the blocking of the downstream MAP kinase pathway, resulting in the inhibition of ERK1 and p38 MAP kinase pathways. This inhibition blocks the migration of endothelial cells.
Recently, another hypothesis has been suggested to explain the function of endostatin as a putative inhibitor of the Wnt signalling pathway (Hanai et al., JCB 158:529, 2002). Wnt signaling is important for the regulation of cell proliferation, differentiation, motility and morphogenesis. Endostatin modulates the Wnt signalling pathway by regulating β-catenin stability via a novel GSK3-independent mechanism. That effect of endostatin on the Wnt signalling pathway triggers the inhibition of endothelial cell migration and induces the entry into the S phase of the cell cycle, which is related to angiogenesis inhibitory activity. Thus, rather than directly affecting the tumor tissue, endostatin indirectly affects the tumor tissue by suppressing new blood vessel construction and blood supply into tumor tissue, which makes it an attractive target for anticancer drug development.
Therefore, a number of clinical approaches have been tried to use endostatin, an anti-angiogenesis agent, for treatment of cancer in humans, but there has not been any positive outcome so far. The in vivo pharmacokinetics of endostatin and the administration mode which has a great impact on efficacy are considered as major obstacles preventing success in clinical trials using endostatin.
According to previous studies, in order for endostatin to be activated in vivo, endostatin must be 1) expressed from a bacterial expression system in a soluble form; 2) capable of being purified in large quantities; 3) capable of being directly administered into the body of the test animal with an injection tool; 4) capable of being maintained at a considerably high in vivo concentration by means of non-continuous direct injection. When such requirements are met, endostatin can function as a critical factor in inducing the apoptosis of cancer cells by inhibiting angiogenesis in tumor tissues.
Meanwhile, small molecules derived from synthetic compounds or natural compounds are capable of being transported into the cells, whereas macromolecules, such as proteins, peptides, and nucleic acids, cannot. It is widely understood that macromolecules larger than 500 kDa are incapable of penetrating the plasma membrane, i.e., the lipid bilayer structure, of living cells. In order to overcome this problem, “macromolecule intracellular transduction technology (MITT)” was developed (Jo et al., Nat. Biotech. 19: 929-33, 2001), which allows the delivery of therapeutically effective macromolecules into cells, making the development of new drugs using peptides, proteins and genetic materials possible. According to this method, if a target macromolecule is fused to a “hydrophobic macromolecule transduction domain (MTD)” and other cellular delivery regulators, synthesized, expressed, and purified in the form of a recombinant protein, it can penetrate the plasma membrane lipid bilayer of the cells, be accurately delivered to a target site, and then, effectively exhibit its therapeutic effect (U.S. Provisional Patent Application No. 60/887,060; PCT International Publication No. WO 2008/093982). Such MTDs are fused to peptides, proteins, DNA, RNA, synthetic compounds, and the like, facilitating the transport of many impermeable materials into the cells.
Accordingly, the inventors of the present invention have developed an endostatin recombinant protein (CP-endostatin) imparted with cell permeability by fusing the angiogenesis inhibitor endostatin to a MTD and found that this recombinant protein effectively delivered a large amount of endostatin into a cell in vivo as well as in vitro to suppress the formation of microvessels and can be used in the treatment of various cancers in humans.